Defining gene regulatory networks driving cortical evolution and brain development

定义驱动皮质进化和大脑发育的基因调控网络

• 批准号: 10039955
• 负责人: Luis de la Torre-Ubieta
• 金额: $ 75.95万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10039955
• 关键词: ATAC-seq; Adult; Affect; Atlases; Automobile Driving; Biochemical; Biological; Biological Assay; Brain; CISH gene; CRISPR interference; CRISPR/Cas technology; Cell Nucleus; Cells; ChIP-seq; Chromatin; Code; Cognition; Cognitive; Data; Development; Disease; Engineering; Enhancers; Equilibrium; Evolution; Excision; Experimental Models; FGFR2 gene; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic Determinism; Genetic Variation; Genome; Genomics; Human; Impairment; LYN gene; Length; Libraries; Macaca; Maps; Mass Spectrum Analysis; Molecular; Morphogenesis; Mus; Neocortex; Neuroglia; Neurons; Nuclear; Nucleic Acid Regulatory Sequences; Outcome; Phenotype; Pregnancy; Protein Isoforms; Proteins; Proteomics; Radial; Reading; Regulator Genes; Regulatory Element; Research Personnel; Resolution; Risk; Role; Specificity; Structure; Technology; Testing; Therapeutic; Tissues; Untranslated RNA; Validation; Work; brain size; cell type; falls; genetic variant; genomic variation; insight; nerve stem cell; neurogenesis; neuropsychiatric disorder; next generation sequencing; novel; progenitor; programs; promoter; social; spatiotemporal; transcription factor; transcriptome

PROJECT SUMMARY/ABSTRACT Neuropsychiatric disorders often affect our most distinguishing cognitive and social capabilities, which are thought to have developed as a result of the expansion of the human neocortex. The unique mechanisms orchestrating cortical neurogenesis and differentiation in the developing human neocortex forming the basis of this expansion are beginning to be described. However, although genetic variation in non-coding gene-regulatory regions, rather than in protein coding genes, drives these evolutionary changes, the cis gene regulatory elements (GREs), including promoters and enhancers, and the transcription factors (TFs) governing cortical neurogenesis remain to be characterized. To begin to investigate this understudied mechanism, we and others have leveraged next generation sequencing approaches to profile chromatin accessibility and interaction in parallel with gene expression to create GRE maps of varying levels of spatiotemporal specificity. We previously identified thousands of developmentally dynamic GREs and their putative gene targets by contrasting GRE activity in progenitor versus neuron-enriched laminae of mid-gestation human neocortex, and functionally validated the role of select GREs in cortical neurogenesis using primary human neural progenitor cells. Further, we found that human-gained enhancers (HGEs), a subset of GREs more active in the human than the macaque or mouse neocortex, regulate genes enriched in outer radial glia (oRG), a neural progenitor with prominent roles in cortical gyrification. This work supports the hypothesis that human developmentally dynamic GREs and HGEs direct gene expression programs controlling the proliferation and differentiation of progenitor pools key to cortical expansion. In this proposal, we seek to test this hypothesis and move from a tissue- and gene-level resolution atlas to a cellular- and gene isoform-level resolution atlas. We will perform single nucleus ATAC-seq to identify cell-specific GREs and leverage a novel single-cell isoform sequencing (scIso-seq) technology to investigate a previously understudied mechanism of gene regulation – alternative promoter usage. This new atlas will inform our work to functionally define the GREs impacting cortical neurogenesis at scale using CRISPR interference (CRISPRi) libraries containing capture tags enabling simultaneous reading of transcriptome and sgRNA at the single-cell level. Finally, we will define and characterize the TFs directing the balance of proliferation versus differentiation of progenitors. These results will enable us identify the cellular basis of genomic variation causing risk for neuropsychiatric disease, and influencing cognition and brain structure. Together this work will create a robust single cell-resolution functional annotation of non-coding GREs and TFs acting in developing human neocortex and elucidate evolutionary mechanisms driving cortical expansion. Broadly, this work will provide a blueprint for scalable approaches to study non-coding genetic variation and cellular diversity.

项目概要/摘要 神经精神疾病通常会影响我们最显着的认知和社交能力，这些能力是 被认为是由于人类新皮质扩张而发展起来的。独特的机制 在发育中的人类新皮质中协调皮质神经发生和分化，形成 这种扩展正在开始被描述。然而，尽管非编码基因调控的遗传变异 驱动这些进化变化的是顺式基因调控元件，而不是蛋白质编码基因中的区域 （GRE），包括启动子和增强子，以及控制皮质神经发生的转录因子（TF） 仍有待表征。为了开始研究这种未被充分研究的机制，我们和其他人利用了 下一代测序方法可与基因并行分析染色质可及性和相互作用 表达式来创建不同级别的时空特异性的 GRE 地图。我们之前确定了 通过对比 GRE 活动，数以千计的发育动态 GRE 及其假定的基因目标 祖细胞与妊娠中期人类新皮质的神经元富集层，并在功能上验证 使用原代人神经祖细胞选择 GRE 在皮质神经发生中的作用。进一步，我们发现 人类获得的增强子 (HGE)，是 GRE 的一个子集，在人类中比猕猴或小鼠更活跃 新皮质，调节富含外放射状胶质细胞（oRG）的基因，外放射状胶质细胞是一种在皮质中发挥重要作用的神经祖细胞 旋回。这项工作支持这样的假设：人类发育动态的 GRE 和 HGE 直接 控制皮层关键祖细胞库增殖和分化的基因表达程序 扩张。在这个提案中，我们试图测试这个假设，并从组织和基因水平的分辨率转向 图谱到细胞和基因亚型水平的分辨率图谱。我们将进行单核 ATAC-seq 来识别 细胞特异性 GRE 并利用新型单细胞异构体测序 (scIso-seq) 技术来研究 之前研究过的基因调控机制——替代启动子的使用。这本新地图集将告知 我们的工作是使用 CRISPR 干扰功能性地定义大规模影响皮质神经发生的 GRE (CRISPRi) 文库包含捕获标签，可同时读取转录组和 sgRNA 单细胞水平。最后，我们将定义和描述指导扩散与扩散平衡的 TF。 祖细胞的分化。这些结果将使我们能够确定导致基因组变异的细胞基础 神经精神疾病的风险，并影响认知和大脑结构。这项工作将共同创造一个 对人类发育中的非编码 GRE 和 TF 进行强大的单细胞分辨率功能注释 新皮质并阐明驱动皮质扩张的进化机制。总的来说，这项工作将提供 研究非编码遗传变异和细胞多样性的可扩展方法的蓝图。